Unidad Manejadora de Aire (UMA)

Initially the effects of the RNA polymerase inhibitors actinomycin D, rifampicin, streptolydigin and tagetitoxin on growth of C. reinhardtii cells in liquid culture was tested. The effects on growth in both heterotrophic (TAP in the light) and phototrophic (HSM in the light) conditions were studied. If PEP activity is dispensable for cell viability, but essential for phototrophic growth, as in higher plants, then the cells should grow in heterotrophic conditions, but not in phototrophic conditions. Streptolydigin at lOOpg/ml and tagetitoxin at 0.1 pM have no effect on W T or cell wall deficient C. reinhardtii. As these inhibitors specifically inhibit PEP, the most likely explanations for this is that the concentrations used in these experiments are too low to have an inhibitory effect in vivo or that the inhibitors can not penetrate the cell or the chloroplast. Rifampicin, however, inhibits cell growth in both heterotrophic and phototrophic conditions at lOOpg/ml. This also suggests that PEP is essential for cell viability as NEP is not sensitive to rifampicin.

The fluorescence emission spectra allows the effect of rifampicin on PSI, PSIl and the LHCs to be studied. The amount of PSIl relative to PSI in the rifampicin treated sample is lower than in the untreated sample, as measured

_________________________________________________________________ Chapter 3 by the intensities of the 680nm and 720nm peaks. PSIl turns over faster than RSI, so blocking transcription would have an immediate effect on levels of PSIl, whereas the levels of PSI would drop more slowly. This also explains the drop in levels of PSIl relative to LHC, as measured by the shift of the 680nm peak to a lower wavelength. Within PSIl the D1 polypeptide, encoded by the psbA gene, is turned over most rapidly, so it seems likely that rifampicin has an effect on the transcription of psbA. Although PSI turns over more slowly than PSIl (Aro et al., 1993) rifampicin has still had an effect as the 720nm peak is also shifted to a lower wavelength. This shift is characteristic of structural changes in PSI, and could be the result of rifampicin blocking transcription of a chloroplast encoded structural subunit of PSI which is turned over more quickly than the other subunits. A possible candidate for this is the protein encoded by psaC which has two iron-sulphur (Fe-S) centres and is, therefore, very sensitive to O2, which can oxidise the

Fe-S centres. The PsaC protein is probably turned over more rapidly than the other proteins in PSI due to damage to the Fe-S centres. These effects are different to those observed in the sample grown under an orange filter where there is more PSIl relative to PSI as measured by the relative intensities of the 680nm and 720nm peaks. PSIl uses the shorter wavelengths of light that are blocked out by the orange filter so more PSIl is produced to compensate for this. The effects observed in the rifampicin treated sample are a result of the inhibition of the RNA polymerase, rather than an effect of the light.

Finally the effect of rifampicin on transcription was studied. A transcription assay was developed in which RNA transcripts are pulse labelled with ^^P- UTP in vivo. The RNA transcripts are then extracted from the cells and used to probe a Southern blot which has a variety of chloroplast, mitochondrial and nuclear gene fragments immobilised to it. The transcripts will hybridise to the gene fragments and those which have been transcribed during the pulse labelling can be visualised by autoradiography. This was carried out in the

unaffected by rifampicin as a transcript can be detected for the nuclear rRNA gene. The effect of rifampicin on transcription of the mitochondrial genome could not be determined as this assay is not sensitive enough to detect the cox1 transcript, encoding a subunit of cytochrome c oxidase, in the presence or absence of rifampicin. The assay was not sensitive enough to detect the chloroplast rpoC2 (encoding a subunit of PEP) cemA (encoding an envelope membrane protein) or orf2971 (encoding a putative nucleoid binding protein) transcripts. This is unsurprising as neither rp o C l nor or/2971 can be detected by northern analysis and are poorly expressed genes. The chloroplast rbcL

(encoding a subunit of Rubisco), atpA (encoding a subunit of ATP synthase),

psaA (encoding a subunit of PSI), psbC (encoding a subunit of PSIl) and rrnS

(encoding a rRNA) transcripts could be detected. Only the transcript of the

rniS gene could be detected in the presence of rifampicin at levels comparable to the

untreated control.

The effect of rifampicin on transcriptionally active extracts from higher plant and algal chloroplasts has been tested and given conflicting results. Bottomley and co-workers found that rifampicin had no effect on chloroplast transcription in pea, maize, radish and spinach neither in vivo nor in vitro

(Bottomley et al., 1971) whereas Surzycki found that the chloroplast RNA polymerase of C. reinhardtii was inhibited in vivo and in vitro by this drug (Surzycki, 1969). Despite reports that the chloroplast RNA polymerase of C.

reinhardtii is completely inhibited by rifampicin as measured by incorporation of NTPs into chloroplast RNA (Guertin & Bellemare, 1979) Jahn found that the C. reinhardtii ch\orop\asi tRNA^'" gene was transcribed in the presence of rifampicin (Jahn, 1992). In mustard distinct rifampicin sensitive and insensitive RNA polymerase activities were identified in etioplasts and chloroplasts. In etioplasts the rifampicin sensitive B enzyme predominates, whereas in chloroplasts the rifampicin insensitive A enzyme is the principle activity (Pfannschmidt & Link, 1994). Both A and B enzymes transcribe all gene classes in vitro and rifampicin inhibits transcription of all gene classes by the B enzyme. However, transcript levels of psbA and rbcL in vivo are reduced in seedlings grown in rifampicin whereas transcript levels of rps^6,

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tmG, rrn and rpoB are unaffected (Pfannschmidt & Link, 1997). It has recently been shown that the A and B enzymes are, in fact, structurally related and can be interconverted by phosphorylation of the B enzyme or dephosphorylation of the A enzyme (Pfannschmidt et a i, 2000).

It was hoped that these inhibitor studies would provide evidence for a second RNA polymerase activity in C. reinhardtii chloroplasts. The clear demonstration of a rifampicin insensitive RNA polymerase activity in C.

reinhardtii chloroplasts which specifically transcribes housekeeping genes would indicate the presence of a NEP activity. However, the inhibitory effect of rifampicin on PEP is poorly understood and it appears that PEP exists in rifampicin sensitive and insensitive forms. It is possible that the rrnS gene is being transcribed by a non-phosphorylated form of PEP. To overcome this a more specific PEP inhibitor, such as tagetitoxin, could be used in an in vivo

transcription assay if an inhibitory effect can be seen on cell growth. A higher concentration of tagetitoxin, for example, may have an inhibitory effect on growth of C. reinhardtii.